How do I Develop a Sample Prep Method?

This is a question that I am often asked by very smart people that just haven’t had time to learn the process. An optimized sample preparation method is critical for an accurate, specific, robust clinical LC-MS/MS assay.  Once the LC and MS/MS conditions for the compounds of interest are optimized, and method parameters like desired LOQ/LOD, analytical measurement range and specimen volume used for analysis have been established, sample prep method development can begin.  A good sample prep method can improve accuracy and precision, provide longer LC column lifetime, and keep your LC-MS/MS system clean.

There are many things to consider when developing a sample prep method:  Do I want minimal clean up or a more specific extraction technique?  What interferences are specific to my matrix that I need to be concerned about?  What type of sample pretreatment do I need to get the best results from my sample prep?  I use a system called “plate mapping” to design my experiments.  It allows me to systematically test different ranges or variables to optimize an extraction, scout a single variable or many at a time, and save time by generating data on multiple variables at once.  Let’s look at a quick example: A basic in plasma.  The drug has a molecular weight of 300 amu, and has an amine attached to aromatic ring.  The logP is 4.5, and the pKa of the amine is 8.2.  Desired LOQ is 2 ng/mL, and desired specimen volume is 100 – 200 µL of plasma, and I want to do minimal clean up.

Minimal cleanup can be accomplished with a phospholipid depletion/protein removal plate like Biotage ISOLUTE PLD+.  According to the package directions, we add acetonitrile to each well of the plate, and then add sample, mix, and elute.  Variables are the addition of 1% formic acid to the acetonitrile, and to vary the acetonitrile:specimen ratio.  I would design a plate map that looks like this:

Samples are run in triplicate at 5-10 times the desired LOQ.  In this experiment, we are evaluating 2 different specimen volumes (100 and 200 µL), three different solvent:specimen ratios (3:1, 4:1 and 6:1) and samples analyzed with and without formic acid. I also analyze an extraction blank, and a negative control (drug free plasma) for each set of conditions.  I would also analyze a no matrix control (unextracted control) at the same concentration to assess process efficiency.  The best option is determined by looking at average area counts and signal:noise for each set of conditions.

If you’d like to learn more, Biotage is presenting a one day seminar in April and May on how to do systematic sample prep method development for clinical LC-MS/MS assays.  Details can be found here:

Join us!




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